Anti myc tag mouse monoclonal antibody

circRNAs bound to HuD were isolated from the striatum of HuD-OE mice by RNA immunoprecipitation (RIP) using Dynabeads^® (Thermo Fisher Scientific) coated with mouse monoclonal anti-myc tag antibody (9B11; Cell Signaling Technology Inc.) specifically recognizing myc-tagged HuD transgenic protein expressed in HuD-OE as described before (Bolognani et al., 2010 (link); Zimmerman et al., 2020 (link)). Controls for RIP assays were performed using either non-immune IgG and HuD-OE tissue or the myc-tag antibody and wild type (WT) tissue. Aliquots for both RNA and proteins were taken before (input) and after the IP and circRNAs were quantitated using circRNAs arrays as described below. Immunoprecipitated RNA was extracted with Trizol^® (Invitrogen) according to manufacturer instructions and subjected to either circRNA array or qRT-PCR as described below.

Mouse monoclonal anti-BACE1 antibody was from Chemicon. Rabbit polyclonal anti-BACE1 antibody was from Calbiochem (USA). Mouse monoclonal anti-clathrin heavy chain and rabbit polyclonal anti-Golgi-localized, gamma adaptin ear-containing, ARF-binding protein 1 (GGA1), goat polyclonal anti-HA tag antibodies were from Abcam (UK). Mouse monoclonal anti-Adaptin α antibody recognizing AP-2 α-subunit was from BD Transduction Laboratories (USA). Mouse monoclonal anti-β-actin, rabbit polyclonal anti-APP C-terminus, mouse monoclonal anti-Aβ 6E10, rabbit polyclonal anti-HA tag antibodies were from SIGMA (USA). Rabbit polyclonal anti-myc tag antibody was from Millipore (USA). Mouse monoclonal anti-myc tag antibody was from Cell Signaling Technology (USA). Mouse monoclonal anti-transferrin receptor (TfR) antibody was from Invitrogen (USA). Goat polyclonal anti-Early Endosome Antigen 1 (EEA1), goat polyclonal anti-Lysosome-associated membrane protein 2 (Lamp2), normal anti-mouse IgG and normal anti-rabbit IgG were from Santa Cruz Biotechnology, Inc (USA). Alexa Fluor 546 mouse IgG, Alexa Fluor 488 mouse IgG, Alexa Fluor 405 mouse IgG, Alexa Fluor 546 rabbit IgG, Alexa Fluor 488 rabbit IgG and Alexa Fluor 488 goat IgG antibodies were obtained from Molecular Probes (USA).

For Western blotting, the samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions and transferred onto Hybond-ECL (GE Healthcare). The recombinant proteins were detected using mouse monoclonal anti-Myc tag antibody (Cell Signaling Technology) (1:500) as primary antibody and HRP-conjugated rabbit anti-mouse IgG antibody (Thermo Fisher Scientific) (1:1,000) as secondary antibody. Immunoreactive bands were visualized using an enhanced chemiluminescence detection system (PerkinElmer) as described (Yamashita et al., 2002 (link)).
To detect Cebelin by immunocytochemical analysis, mouse monoclonal anti-Myc tag antibody and FITC-conjugated goat anti-mouse IgG (Sigma-Aldrich) were used as primary and secondary antibodies, respectively. To detect Mannosidase II, EEA1, and GRP78, rabbit anti-Mannosidase II antibody, anti-EEA1 antibody, and anti-GRP78 antibody (Abcam) and TRITC-conjugated goat anti-rabbit antibody (Sigma-Aldrich) were used as primary and secondary antibodies, respectively.

HeLa cells were cultured in HyClone™ High Glucose Modified Eagles medium (GE Healthcare Life Sciences), supplemented with 10% fetal bovine serum, 4 mM L-Glutamine, 4500 mg/L Glucose, Sodium Pyruvate, and 100 U/ml Penicillin/Streptomycin. HeLa cells were incubated in a 37C° and 5% CO2 humidified incubator. When cells reached 60–80% confluency, they were seeded on sterile cover slips in 24-well culture plates. After 24 h of incubation, cells were transfected with either wild-type or mutant plasmids, and each was co-transfected with GFP-H-Ras plasmids using FuGENE HD (Promega). The GFP-H-Ras was used as a plasma membrane marker. 0.5 μg of each plasmid was incubated with Gibco™ Opti-MEM™ I Reduced Serum Media (Opti-MEM), followed by (3:1) ratio of (FuGENE:DNA), respectively. After 24 h of incubation at 37C^o, cells were washed three times with PBS, fixed with absolute methanol then blocked with 2% bovine serum albumin (BSA) (Sigma) for 1 hour. Finally, cells were stained with mouse-anti-Myc-tag monoclonal antibody (dilution: 1:200; Cell Signaling Technology) for 1 hour. Followed by three times wash with PBS and incubation with anti-mouse IgG Fab2 Alexa Flour 555 antibody (dilution: 1:200; Cell Signaling Technology). Images were acquired using Nikon confocal Eclipse 80I microscope (Japan).